Trichomonas vaginalis is a sexually transmitted protist causing serious health consequences for women. Trichomonosis is a co-factor in HIV transmission and contributes to preterm labor, cervical cancer and atypical pelvic inflammatory disease. Incidence and prevalence rates of this disease are especially high among minorities, contributing to health disparities. Despite this public health impact, fundamental aspects of trichomonad cell and molecular biology are unknown. Our long-term goal is to understand the molecular basis of adherence of trichomonads to vaginal epithelial cells. The rationale is that molecular dissection of this fundamental process of virulence will provide for effective means of control of this infectious disease. The objective of this work is to perform genetic analysis of T. vaginalis adherence mediated by the surface protein AP65, a protein with sequence identity to decarboxylating malic enzyme of the metabolic hydrogenosome organelle. The functional diversity and differential compartmentalization of a metabolic enzyme to mediate adherence is a unique adaptation by this parasite to colonizing the urogenital tract. The hypothesis is that contact with host cells and iron initiate a signal transduction cascade leading to phosphorylation and subsequent mobilization and surface placement of AP65, which allows for interaction of AP65 with a specific protein receptor on the host cell. Further, there is a quantitative relationship between the amount of AP65 on the parasite and the amount of available receptors on host cells that determines the level of adherence. To test the hypothesis, three specific aims are proposed: Aim 1 will silence and modulate expression of the AP65 adhesin through antisense RNA to provide genetic evidence for a role of AP65 in adherence. Aim 2 will express an epitope-tagged AP65 to study compartmentalization and surface targeting in response to iron levels. Aim 3 will characterize the role of phosphorylation of AP65 as a signal for surface placement. The aims will utilize episomally-expressed constructs of mutated phosphorylation sites and/or constructs with mutated regions within AP65 essential for surface targeting. The use of antisense RNA inhibition, the demonstration of signaling from host to parasite for protein surface targeting, and establishment of a stable transfection system for heterologous expression in trichomonads represent innovative aspects of research for studying the biology of Trichomonas organisms.